This invention relates to typing hepatitis C viruses (HCV). In particular, this invention relates to a method of typing HCV using novel type specific polypeptides.
Viral hepatitis is known to be caused by five different viruses known as hepatitis A,B,C, D and E. HAV is an RNA virus and does not lead to long-term clinical symptoms. HBV is a DNA virus. HDV is a dependent virus that is unable to infect cells in the absence of HBV. HEV is a water-borne virus. HCV was first identified and characterized as a cause of non-A, non-B hepatitis (NANBH). Houghton et al., EPO Pub. No. 388,232. This led to the disclosure of a number of general and specific polypeptides useful as immunological reagents in identifying HCV. See, e. q., Choo et al. (1989) Science, 244:359-362; Kuo et al. (1989) Science, 244:362-364; and Houghton et al. (1991) Hepatology, 14:381-388. HCV is the major cause of blood transfusion-related hepatitis.
The prototype isolate of HCV was characterized EP Publication Nos. 318,216 and 388,232. As used herein, the term xe2x80x9cHCVxe2x80x9d includes newly isolated NANBH viral species. The term xe2x80x9cHCV-1xe2x80x9d refers to the virus described in the above-mentioned publications.
Since the initial identification of HCV, at least six different viral types have been identified and designated HCV-1 to HCV-6. Cha et al. (1992) Proc. Natl. Acad. Sci. USA, 89:7144-7148. Within these types are numerous subtypes. The type of virus with which a patient is infected may affect the clinical prognosis and also response to various treatments. Yoshioka et al. (1992) Hepatology, 16:293-299. In light of the fact that the most serious clinical outcome of HCV infection is hepatocellular carcinoma, it would be useful to be able to determine with which type or types of HCV a patient is infected.
The method currently in use to determine virus type is genotyping; that is, isolation of viral RNA and determination of the sequence of various segments by polymerase chain reaction (PCR). Not only is this method laborious and time consuming but it is not suitable for use on samples that have been stored under conditions that do not allow for preservation of RNA or samples from patients that do not have sufficient viral titer. It would be useful to have a method for typing HCV by immunoanalysis or serotyping.
The current method for screening blood and diagnosing patients is an immunoassay. The immunoassay utilizes an antigen from HCV-1 which contains a sufficient number of common epitopes to detect antibodies to other types of HCV. The immunoassay does not distinguish between infections by different types of HCV.
The present invention includes compositions and methods for typing of HCVs by genotype and serotype. The compositions include type specific epitopes, type-cluster specific epitopes, nucleic acids encoding the epitopes for use as probes and nucleic acids complementary to the regions flanking those encoding the epitopes for use as primers.
One aspect of the invention is a method for typing HCV comprising the steps of providing an antibody-containing sample from an individual; contacting the sample with a type specific epitope or type-cluster specific epitopes under conditions which permit antigen-antibody binding; and determining whether antibodies in the sample bind to the epitope.
Another aspect of the invention relates to a method for typing HCV comprising the steps of providing an antibody-containing sample from an individual; contacting the sample with a first type specific epitope or type-cluster specific epitope under conditions which permit antigen-antibody binding; contacting the sample with a second type specific epitope or type-cluster specific epitope under conditions which permit antigen-antibody binding; and determining whether antibodies in the sample bind to either the first or second epitope.
Another aspect of the invention relates to polypeptides containing type specific epitopes or type-cluster specific epitopes. The polypeptides are derived from three different regions of the HCV genome. One set of polypeptides includes type specific epitope or type-cluster specific epitopes obtained from the HCV core region. This first set is found between amino acid residues sixty-seven and eighty-four of HCV-1 and homologous regions of other types of HCV. As used herein, the amino acid residue abbreviations are as follows: A, alanine; I, isoleucine; L, leucine; M, methionine; F, phenylalanine; P, proline; W, tryptophan; V, valine; N, asparagine; C, cysteine; Q, glutamine;; G, glycine; S, serine; T, threonine; Y, tyrosine; R, arginine; H, histidine; K, lysine; D, aspartic acid; and E, glutamic acid.
The particular amino acid residue sequences derived from the core region and subtypes from which they are derived are as follows:
1. PEGRTWAQ (SEQ ID NO:3), subtype 1a or 1b.
2. STGKSWGK (SEQ ID NO:4), subtype 2a or 2b.
3. SEGRSWAQ (SEQ ID NO:5), subtype 3a or 4.
Another set of polypeptides includes a type specific epitope obtained from the HCV non-structural region 4 (NS4). This second set is found between amino acid residues 1689-1718 of HCV-1 and homologous regions of other types of HCV.
The particular amino acid residue sequences and types or subtypes from which they are derived are as follows:
1. CSQHLPY (SEQ ID NO:6), subtype 1a.
2. CASHLPY (SEQ ID NO:7), subtype 1b.
3. CASRAAL (SEQ ID NO:8), subtype 2a or 2b.
4. CASKAAL (SEQ ID NO:23)
Another set of polypeptides includes type specific epitope or type-cluster specific epitopes obtained from the non-structural region 5 (NS5) of a hepatitis C virus. This set is found between amino acid residues 2281-2313 of HCV-1 and homologous regions of other types of hepatitis C virus.
The particular amino acid residue sequences and, types or subtypes from which they are derived are as follows:
1. PDYEPPVVHG (SEQ ID NO:9), subtype 1a.
2. PDYVPPVVHG (SEQ ID NO:10), subtype 1b.
3. PDYQPATVAG (SEQ ID NO:11), subtype 2a.
4. PGYEPPTVLG (SEQ ID NO:12), subtype 2b.
5. FAQASPVW (SEQ ID NO:13), subtype 1a.
6. FPPQALPIW (SEQ ID NO:14), subtype 1b.
7. FPPQALPAW (SEQ ID NO:15), subtype 2a.
8. FPPQALPPW (SEQ ID NO:16), subtype 2b.
Another aspect of the invention includes nucleic acid molecules encoding the amino acid residue sequences of the type specific and type-cluster epitopes described. These nucleic acid molecules are useful as probes for instance in Southern blots or other DNA recognition assays such as the capture assay described in U.S. Pat. Nos. 4,868,105; and 5,124,246.
Another aspect of the invention includes nucleic acid molecules complementary to the nucleic acid sequences flanking regions encoding the type specific and type-cluster specific epitopes. Such nucleic acid molecules are useful in performing PCR to determine the genotype of a particular HCV.